Role of activated macrophages in resistance to systemic candidosis

To evaluate further the contribution of activated macrophages in resistance, the course of systemic candidosis was assayed in beige and NLM mice that had been previously infected with Mycobacterium bovis (BCG). Four weeks following BCG infection, mice were inoculated intravenously with 1 x 10(4) viable Candida albicans. At various times thereafter, the number of C. albicans colony-forming units in the livers, spleens, and kidneys was determined. The average number of CFU recovered from the kidneys of NLM mice decreased throughout the assay and was comparable in both BCG-treated and control mice. In contrast, the number of CFU cultured from the kidneys of untreated control beige mice progressively increased throughout the assay period. This profile of renal susceptibility was not appreciably altered in BCG-treated beige mice. However, fewer (10- to 100-fold) CFU were cultured from the livers and spleens of BCG-treated beige and NLM mice than from untreated controls. These results support the hypothesis that in the absence of functional polymorphonuclear leukocytes, activated macrophages represent a means to control the proliferation of C. albicans.     

Systemic candidosis in silica-treated athymic and euthymic mice.

Intravenous silica injections were used to assess the role of macrophages in the resistance of BALB/c nude and euthymic mice to systemic candidosis. CFU of Candida albicans in the kidneys, livers, and spleens of saline- or silica-treated mice were enumerated at various times after inoculation with 10(4) viable yeast cells. The number of C. albicans organisms recovered from the kidneys of silica-treated euthymic mice was similar to the number recovered from saline-treated controls during the first 3 days of infection; however, at every assay period thereafter, the number of organisms recovered from the kidneys of silica-treated mice was dramatically reduced (100- to 1,000-fold). Conversely, silica-treated nude mice were no more susceptible to systemic candidosis than were saline-injected nude mice. Silica treatment did not alter the ability of treated or control mice to clear C. albicans from the liver and spleen. These results demonstrate that macrophages play an important role in susceptibility to Candida infections.     

Systemic and gastrointestinal candidiasis of infant mice after intragastric challenge.

Systemic and gastrointestinal infection can be established in infant mice after intragastric challenge with Candida albicans. Differences in virulence of the six strains tested were noted. As early as 3 h after infection, some but not all livers, spleens, and kidneys contained C. albicans, but the peak number of colony-forming units in these organs was seen at 6 h. The early colonization of the organs could not be attributed to aspiration of the inoculum since about 90% of lungs and livers tested yielded no colony-forming units at 10 to 15 min postinfection. In animals with systemic infections, lungs, livers, kidneys, and spleens showed similar numbers of colony-forming units within the organs during the first 6 h postinfection- and then the number declined progressively up to 72 h. The gastrointestinal tract was colonized throughout a 20-day period of study. Counts made at intervals beyond day 1 yielded between 10(5) and 10(6) colony-forming units in the stomach, ileum, and cecum. Preparatory techniques for scanning electron microscopy preserved the yeast, intestinal mucus layer, and epithelial surface and made it possible to visualize the association between the pathogen and host tissues within the digestive tract.     

Systemic Coccidioides immitis infection in nude and beige mice.

The course of experimental systemic Coccidioides immitis infection was assessed quantitatively and histologically in beige mice, congenitally athymic nude mice, and their respective normal counterparts. After intravenous inoculation with 50 arthroconidia, the number of viable C. immitis cultured from the spleens, livers, and lungs progressively increased throughout the assay in the organs of all mice. During the first 2 weeks of infection, significantly greater numbers of CFU were recovered from the spleens and livers, but not the lungs, of nude mice than from the respective organs of their phenotypically normal littermates. Significantly greater numbers of CFU were cultured from the lungs and spleens of beige mice compared with the number recovered from their functionally normal littermates. After intranasal inoculation, extrapulmonary dissemination of C. immitis occurred at an equal rate and resulted in similar organ burdens in nude mice and their normal littermates. Histological examination of infected tissues revealed a characteristic mixed inflammatory cell infiltrate in euthymic mice; the response in nude mice was less severe, consisting predominantly, if not solely, of granulocytes. In addition, in tissue sections from nude mice, but not in those from their euthymic counterparts, mature spherules were frequently observed to be devoid of an associated inflammatory response. The inflammatory lesion in beige mice contained a predominance of mononuclear cells, whereas their littermates responded with a typical mixed granulomatous infiltrate. Collectively, these results provide evidence supporting the hypothesis that resistance to C. immitis infection involves two primary cell populations, one under the direct influence of T-cells and the other independent of T-lymphocytes.     

Role of natural killer cells in resistance to systemic cryptococcosis

These studies demonstrate that Cryptococcus neoformans infection induced a dose-dependent augmentation of splenic natural killer (NK) cell activity by bg/+, but not bg/bg mice. To directly assess the role of NK cells in resistance to C. neoformans, bg/+ and bg/bg mice were treated with anti-NK-1.1 monoclonal antibody (mAb). Anti-NK-1.1-treatment abrogated the augmented NK cell activity observed during C. neoformans infection in bg/+ mice. Anti-NK-1.1-treated bg/+ mice had higher C. neoformans colony forming units (CFU) in their lungs on days 3 and 7 after intravenous (i.v.) challenge than control bg/+ mice. Moreover, the number of C. neoformans CFU in the lungs of anti-NK-1.1-treated bg/+ mice on days 3 and 7 were similar to those observed for infected bg/bg mice. By day 14, however, no differences in C. neoformans CFU were evident in the lungs of anti-NK-1.1-treated and control bg/+ mice. Anti-NK-1.1-treatment did not alter either the growth of C. neoformans in the spleens, livers, kidneys, or brain of bg/+ mice or the susceptibility of bg/bg mice to systemic cryptococcosis. These studies suggest that NK cells do not play a role in resistance to systemic cryptococcosis in the spleen, but do appear to play an early, but transient role in resistance to C. neoformans in the lungs. Overall, congenital defects in polymorphonuclear neutrophils (PMNs) and macrophages (M phi s), in addition to defects in NK cells, contribute to the enhanced susceptibility of bg/bg mice to systemic cryptococcosis.     

Clearance of Cryptococcus neoformans from immunologically suppressed mice.

To assess the effects of cryptococcal antigen-induced immunosuppression on a Cryptococcus neoformans infection, CBA/J mice were injected intravenously with saline or suppressive doses of cryptococcal antigen (CneF) at weekly intervals and were then infected with viable C. neoformans cells. By the second week after infection, the cryptococcal antigen-injected mice had suppressed anticryptococcal delayed-type hypersensitivity (DTH) responses compared with the responses of the saline-treated, infected control mice. In addition, the immunosuppressed mice had higher numbers of cryptococcal CFU cultured from their lungs, livers, spleens, lymph nodes, and brains than did the control animals. A direct correlation of suppression of the anticryptococcal DTH response and reduced clearance of cryptococci from tissues was also observed after mice were given a single intravenous injection of CneF and infected. To determine whether or not the cryptococcal antigen was specifically reducing the clearance of C. neoformans or had a more generalized effect, mice were injected with saline or suppressive doses of CneF, infected with Listeria monocytogenes, and then followed daily for 7 days for the clearance of L. monocytogenes from spleens and on day 7 for DTH reactivity to Listeria antigen. There were no differences between the saline- and CneF-treated mice with respect to anti-Listeria DTH responses or clearance of L. monocytogenes from spleens, indicating that CneF was not altering natural resistance mechanisms responsible for early clearance of L. monocytogenes, nor was the CneF influencing the induction of the acquired immune response which was responsible for the late clearance of the bacteria. Together, these data indicate that the specific suppression of this cell-mediated immune response induced by cryptococcal antigen reduces the ability of the animals to eliminate the homologous organism (C. neoformans) but not a heterologous infectious agent, such as L. monocytogenes.     

Effect of abrogation of natural killer cell activity on the course of candidiasis induced by intraperitoneal administration and gastrointestinal candidiasis in mice with severe combined immunodeficiency.

Candida albicans CFU per gram of tissue recovered from livers, spleens, and kidneys of 12 severe combined immunodeficiency (scid) and 12 BALB/c mice 5 days after intraperitoneal (i.p.) administration of 10(7) C. albicans cells were not significantly different. Nine scid mice given normal rabbit serum (NRS) as a control and eight scid mice given anti-asialo-GM1 (alpha-ASGM1) had C. albicans CFU per gram recovered from livers and spleens 1 week after i.p. administration of C. albicans that were not significantly different, despite virtual elimination of natural killer (NK) cell activity in mice treated with alpha-ASGM1. At 2 weeks after i.p. administration, despite significantly increased NK cell activity in eight infected NRS-treated scid mice and virtual elimination of NK cell activity by alpha-ASGM1 treatment of eight scid mice, C. albicans CFU per gram recovered from livers and kidneys were not significantly different. At 2 weeks after intragastric administration of 2 x 10(6) C. albicans cells, eight NRS- and eight alpha-ASGM1-treated scid mice had identical proportions colonized with C. albicans and similar C. albicans CFU per gram recovered from feces. There was no evidence of hematogenous dissemination in either group. Similar results were seen 1 week after intragastric administration of 10(7) C. albicans cells. We conclude that NK cell activity is increased by i.p. administration of C. albicans in scid mice, but nontheless, abrogation of NK cell activity is not associated with enhanced susceptibility to candidiasis induced by i.p. administration and also is not associated with enhanced susceptibility to gastrointestinal colonization or hematogenous dissemination after intragastric administration of C. albicans.     

Granuloma formation in severe combined immunodeficient (SCID) mice in response to progressive BCG infection. Tendency not to form granulomas in the lung is associated with faster bacterial growth in this organ.

Intravenous inoculation of Mycobacterium bovis, strain bacillus Calmette-Guerin caused infection in the lungs, livers, and spleens of severe combined immunodeficient (SCID) mice and in the same organs in immunocompetent co-isogenic C.B17 mice. However, whereas infection in the latter mice was stabilized and partly resolved, it was progressive in SCID mice and eventually lethal, with the most rapid bacterial growth occurring in the lungs. Histological examination of infected organs showed that well-developed, compact epithelioid granulomas formed at sites of bacterial multiplication in livers, spleens, and lungs of C.B17 mice. Granulomas also formed in the livers and spleens of SCID mice, despite their inability to generate immunity. However, in the lungs of SCID mice, bacillus Calmette-Guerin was regionally distributed mostly in isolated alveolar macrophages and in aggregates of macrophages resembling small granulomas. The possibility that this tendency not to form granulomas in the lung is the reason for the more rapid growth of bacillus Calmette-Guerin in this organ is discussed.     

Lack of a role for natural killer cells in early control of Brucella abortus 2308 infections in mice.

Studies were conducted to determine if natural killer (NK) cells are important for early control of the virulent strain Brucella abortus 2308 following infection of mice with high or low challenge doses. Splenocytes from C57BL/10 and BALB/c mice that had been infected with the lower dose of B. abortus displayed increased cytotoxicity against YAC-1 cells during the first week after infection, while infection of C57BL/10 mice with the higher challenge dose either did not alter the level of NK cytotoxic activity or decreased it, depending upon the time postinfection. In vivo depletion of NK cells by monoclonal antibody anti-NK1.1 or polyclonal anti-asialoGM1 antiserum did not result in an increase in the number of brucellae recovered from the spleens or livers of the brucella-resistant C57BL/10 mice or from the spleens of the susceptible BALB/c mice during the first week after infection. Treatment of control mice with the NK-reactive antibodies, however, decreased killing of the NK-sensitive target YAC-1, indicating that the NK cell depletion regimes were effective. Our results suggest that NK cells are not crucial for early control of B. abortus 2308 even though they may be activated following infection. Further experiments indicated that treatment of C57BL/10 mice with poly(A:U) did not decrease the number of brucellae recovered from their spleens although it did decrease the CFU in livers of mice infected with the high challenge dose.     

Trypanocidal drugs and the role of kidney forms ofTrypanosoma (Herpetosoma) musculi in immunity of mice against reinfection

Adrenals, hearts, kidneys, livers, lungs, and spleens were removed from C3H/Anf mice which had been inoculated withTrypanosoma (Herpetosoma) musculi and no longer exhibited parasitemias. Imprints of each organ were examined microscopically, and each was homogenized and injected into recipient mice. It was confirmed that trypanosomes could be detected only in the donor kidneys. Lampit or Ethidium treatment eliminated bloodstream and kidney forms when administration was initiated after the development of patent parasitemias. However, mice treated with Lampit on the same day they were inoculated withT. musculi developed parasitemias later than animals injected with drug after parasites had appeared in their blood. Both Lampit and Ethidium depressed antibody production as detected in enzyme-linked immunosorbent assays of antisera from animals having parasitemias at the time of treatment. The elimination of kidney forms by Lampit or Ethidium treatment did not reduce the resistance of mice to reinfection byT. musculi 12 weeks or 15 and 25 weeks, respectively, after the initial inoculation of these animals with the parasites. Kidney forms were not required for the sustained protective immunity of the mice against reinfection during the intervals of these experiments.     

Reactivity patterns of anti-phospholipid antibodies in systemic lupus erythematosus sera in relation to erythrocyte binding and complement activation.

The protective mechanisms associated with resistance to atypical mycobacteria infections are not clear. In an effort to broaden our understanding of the mechanisms involved, susceptible mice were infected with a virulent strain of M. avium and various treatments were applied so as to modify the course of the disease. Treatment with an antiserum against tumour necrosis factor-alpha (TNF-alpha) significantly enhanced the experimental infection, as judged by enumeration of colony-forming units (CFU) in the spleens and livers of infected mice, suggesting a role for TNF-alpha in resistance to M. avium. In other sets of experiments, recombinant cytokines were directly infused into infected mice. Infusion of recombinant interferon-gamma (IFN-gamma) did not modify the experimental infection significantly, and infusion of interleukin-2 was also without effect. Injection of TNF-alpha enhanced resistance in susceptible animals, as seen by a reduction in the viable bacilli recovered from the spleens and livers. In a final set of experiments, we demonstrate that combinations of cytokines may induce strong resistance against M. avium, namely injection of 1 micrograms of interleukin-1 alpha and 1 micrograms of TNF-alpha at 5-day intervals which was seen to eradicate M. avium in both spleens and livers of susceptible BALB/c mice. Overall, our results suggest that induction of protection against M. avium by treatment with cytokines may be feasible, and that TNF-alpha may be a pivotal molecule in resistance to M. avium.     

Evolution of inflammatory response and cellular immune responses in a murine model of disseminated blastomycosis.

A reproducible model of disseminated blastomycosis was established in C57BL/6 mice by intravenous injection of 10(6) yeast-phase Blastomyces dermatiditis organisms. The infection progressed over 5 weeks to involve lungs, brains, superficial fascia, livers, and spleens of mice. By week 5, there was a greater number of organisms in lungs and brains than in livers and spleens. The tissue response in lungs, brains, and livers progressed from acute neutrophilic invasion before week 1 to pyogranuloma formation by week 5. Lymph nodes and spleens were remarkably spared. By week 5, infected mice became anergic to intradermal challenge with both specific Blastomyces antigen and a nonspecific antigen (sheep erythrocytes). At this time, the response to concanavalin A or phytohemagglutinin by splenocytes was markedly less than that of normal controls. Likewise, the plaque-forming cell response to sheep erythrocytes by splenocytes from infected mice was diminished. In coculture studies, splenocytes from 5-week-infected mice reduced the plaque-forming cell response by normal splenocytes. The development of this murine model should prove useful for elucidating the perturbations of immunoregulation associated with disseminated blastomycosis.     

Role of complement C5 and T lymphocytes in pathogenesis of disseminated and mucosal candidiasis in susceptible DBA/2 mice

The aims of the study were to compare the pathogenesis of Candida albicans infection in various organs and anatomical regions of C5-deficient (DBA/2) and C5-sufficient (BALB/c) mice, and to evaluate the importance of complement C5 and T lymphocytes as factors that determine host susceptibility or resistance. The kidneys of DBA/2 mice showed higher colonisation and more severe tissue damage than those of BALB/c, but infection at other sites, including oral and vaginal mucosa, was generally similar in the two strains. Passive transfer of C5-sufficient serum into DBA/2 mice decreased the fungal burden in the kidney, and prolonged survival of the reconstituted animals. Depletion of CD4(+) and/or CD8(+) cells did not exacerbate either systemic or mucosal infection when compared to controls, and passive transfer of splenocytes from infected donors caused only a small and transient reduction in numbers of yeasts recovered from the kidney of sub-lethally infected recipients. It is concluded that the acute susceptibility of the kidneys in this mouse strain is due to C5 deficiency expressed on a susceptible genetic background. T lymphocytes, however, appear to have minimal influence on recovery from systemic infection with this isolate of C. albicans.     

Effect of oral administration of β-D-glucan from Aureobasidium pullulans ADK-34 on Candida and MRSA infections in immunosuppressed mice

We examined the effect of the oral administration of β-D-glucan derived from Aureobasidium pullulans ADK-34 (AP-FBG) on Candida albicans or methicillin-resistant Staphylococcus aureus (MRSA) infection in immunosuppressed mice. Mice pretreated with cyclophosphamide (CY) were intraperitoneally administered AP-FBG for 4 days and then infected with 6×10(4) C. albicans cells. In a preliminary experiment, the survival time of the Candida-infected mice treated with AP-FBG was clearly prolonged. Similarly, the effect of the oral administration of AP-FBG was examined. Mice were orally given 2.5% AP-FBG in feed for 42 days from 14 days prior to 2×10(4) C. albicans cells infection. The survival time of mice treated with AP-FBG was significantly prolonged and the viable cell count in the kidneys of the survivors was significantly decreased at 30 days after infection. The effects of the oral administration of AP-FBG on intestinal MRSA infection were also examined. Mice were given 2.5% AP-FBG orally in feed for 30 days before and after oral MRSA infection and treated with CY 12 days after the infection. The number of viable MRSA cells or the IgA production in feces did not significantly change, while AP-FBG administration seemed to relieve temporally the loss of body weight of mice.Conclusions: These results suggest that oral pre-administration of AP-FBG promoted resistance of CY-treated mice to C. albicans and lessened the weight reduction of CY-mice infected by MRSA.     

Resistance of high and low antibody responder lines of mice to the growth of avirulent (BCG) and virulent (H37Rv) strains of mycobacteria.

The resistance to Mycobacterium bovis (BCG) of lines of mice selected for high (H) or low (L) antibody responsiveness was estimated from the rate of BCG multiplication in the organs. During the first 2 weeks after i.v. infection with 5 X 10(6) CFU, BCG multiplied faster in the spleens of H than of L mice. Afterwards there was a more drastic reduction of viable BCG counts in H mice than in L mice so that the residual BCG counts were significantly lower in H mice than in L mice, not only in the spleen but also in the liver and lungs. On the 14th day of infection, the spleen and liver enlargement and the increase of phagocytic activity were similar in the two lines, suggesting an identical T lymphokine release. In contrast with BCG, during the first 2 weeks after infection with 7 X 10(5) CFU, M. tuberculosis (H37Rv) multiplied in the spleens of L mice at a similar or a slightly faster rate than in the spleens of H mice. On the 4th week, the viable H37Rv counts were reduced in H mice whereas L mice did not survive the infection. In mice vaccinated with BCG 5 months before virulent challenge, the multiplication of H37Rv was inhibited in the H and L lines. The protective effect of BCG is therefore stronger in L mice taking into account their higher innate susceptibility to H37Rv. This might be due to the higher level of living BCG found in L mice at the time of challenge.     

Host-Etiological Agent Interactions in Intranasally and Intraperitoneally Induced Cryptococcosis in Mice

Inbred CBA/J mice were used in developing a defined in vivo model for studying host-parasite relationships in cryptococcosis. Mice were infected either intranasally or intraperitoneally with 10³ viable Cryptococcus neoformans cells. At weekly intervals over a 92-day period, C. neoformans growth profiles in the lungs, spleens, livers, and brains of the infected animals were determined. In addition, humoral and delayed-type hypersensitivity responses and cryptococcal antigen levels were assayed in these mice. Intranasally infected mice developed strong delayed-type hypersensitivity reactions in response to cryptococcal culture filtrate (CneF) antigen, and there was good correlation between acquisition of delayed-type hypersensitivity and the reduction of C. neoformans cell numbers in infected tissues. In contrast, intraperitoneally infected mice displayed greater numbers of C. neoformans cells in tissues and had somewhat suppressed delayed-type hypersensitivity responses to CneF antigen. Anticryptococcal antibodies were not detected in intranasally or intraperitoneally infected mice, but cryptococcal polysaccharide antigen titers were relatively high in both groups. The transfer of sensitized spleen cells from intranasally infected mice to syngeneic naive recipient mice resulted in the transfer of delayed-type hypersensitivity responsiveness to cryptococcal antigen in the recipients. The intranasally induced infection in mice was similar to the naturally acquired infection in humans; therefore we are proposing that this murine-cryptococcosis model would be useful in gaining a greater understanding of host-etiological agent relationships in this disease.     

The effects of protein malnutrition on the pathogenesis of murine cytomegalovirus disease.

BALB/c and CBA mice maintained on low (4%) or normal (18%) protein diets for 2 weeks after weaning were infected with a sublethal dose of murine cytomegalovirus, adjusted in proportion to body weight. Viral replication, histopathological changes and humoral responses to the virus were compared between the dietary groups 2-42 days post infection (p.i.). Higher numbers of viral antigen-positive cells and/or more prominent tissue necrosis were noted in the livers, spleens, hearts, adrenal glands, kidneys and bone marrows of infected protein-deficient mice. These mice also showed a delayed onset of leucocytic exudation in their livers and salivary glands, relative to infected mice on the normal diet. Second peaks of viral replication were detected by plaque assays in livers and spleens from protein-deficient mice and in livers from normal mice 12-18 days p.i., but few antigen-positive cells and no tissue necrosis were observed. Virus also persisted at higher titres in the salivary glands from protein-deficient mice. Although cellular immunity may be defective in these mice, humoral IgG and IgM responses to the virus were not inhibited. The influence of genetic factors on the pathogenesis of murine cytomegalovirus disease in protein-deficient mice is discussed.     

Changes in T-Lymphocyte Subsets in Lungs and Spleens of Mice with Slowly Progressive Primary Mycobacterium tuberculosis Infection: Involvement of Unconventional T-Cell Subsets

Our previous study showed that the cell-activation responses and cytokine-secretion patterns were different in lungs and spleens of mice with slowly progressive primary Mycobacterium tuberculosis infection. The aim of the present study was to characterize the T-cell subsets in lungs and spleens of mice with a similar infection. The percentages of T-cell subsets were determined by flow cytometry and the absolute numbers were calculated. Spleens of infected mice showed a threefold expansion of CD4+ cells but no change in CD8+ cells, whereas lungs had a threefold increase of both subsets. A significant expansion of CD4-CD8-alphabeta+ [double negative (DN)alphabeta+] subsets was observed in the lungs of infected mice compared with uninfected mice. This was not the case in the spleens of infected mice. In infected mice the CD4-CD8- (DN) population preferentially expressed alphabeta-T-cell receptors (TCR) in the lungs but gammadelta-TCR in the spleens. The percentages of many T-cell subsets were significantly higher in the lungs than in the spleens of both uninfected and infected mice. However, the percentages of CD4+ and CD4-CD8+TCR- subsets in the lungs were significantly lower than in the spleens of infected mice. We also observed some previously unreported T-cell subsets: double positive-TCR- (DPTCR-), DPalphabeta+ and DPgammadelta+. So far their functions are unknown.     

Effect of L18-MDP(Ala), a synthetic derivative of muramyl dipeptide, on nonspecific resistance of mice to microbial infections.

By subcutaneous treatment with an aqueous solution of 6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine [6-O-CH3-(CH2)16-CO-MurNAc-L-Ala-D-isoGln] [referred to here as L18-MDP(Ala)], an augmentation of the resistance of mice to Escherichia coli, Pseudomonas aeruginosa. Staphylococcus aureus, and Candida albicans infections was observed, but not to infections with Klebsiella pneumoniae and Listeria monocytogenes. Against E. coli infections, L18-MDP(Ala) was highly protective, irrespective of the administration route. Bacteremia occurring at an early phase of such infections was almost completely prevented by subcutaneous treatment 1 day before infection. Single or multiple doses were also effective against C. albicans infection. The phagocytosis of E. coli by mouse peritoneal polymorphonuclear cells was enhanced by treatment with the adjuvant, and the phagocytosis of K. pneumoniae was also enhanced, but only when the mice were treated either with rabbit normal serum or with a specific immune serum. The growth of the fungus in the kidneys was significantly inhibited, and growth was eliminated from the kidneys by treatment with the adjuvant once a day for 4 consecutive days, starting 1 day before infection. However, no growth suppression of L. monocytogenes in the livers or spleens of infected mice was observed when they were treated with a single dose of the adjuvant. This difference may be ascribed to the differences in the effector mechanisms of defense and to the different degree of augmentation of each defense mechanism by L18-MDP(Ala).